US6603553B1 - Arrangement for detecting fluorescent light from a plurality of sample points - Google Patents

Arrangement for detecting fluorescent light from a plurality of sample points Download PDF

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Publication number
US6603553B1
US6603553B1 US09/681,109 US68110901A US6603553B1 US 6603553 B1 US6603553 B1 US 6603553B1 US 68110901 A US68110901 A US 68110901A US 6603553 B1 US6603553 B1 US 6603553B1
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Prior art keywords
specimen
specimen points
points
fluorescent light
slide
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US09/681,109
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English (en)
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Johann Engelhardt
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Leica Microsystems CMS GmbH
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Leica Microsystems Heidelberg GmbH
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Assigned to LEICA MICROSYSTEMS HEIDELBERG GMBH reassignment LEICA MICROSYSTEMS HEIDELBERG GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ENGELHARDT, JOHANN DR.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G01N21/6454—Individual samples arranged in a regular 2D-array, e.g. multiwell plates using an integrated detector array

Definitions

  • the invention relates to an arrangement for detecting fluorescent light from a plurality of specimen points, in particular from microgene spots or microbiospots, the specimen points being arranged on a slide, having at least one light source for simultaneously illuminating the specimen points with excitation light, and having detection means, comprising detection elements, for simultaneously detecting the fluorescent light from the individual specimen points.
  • Such arrangements are used, for example, for investigations in molecular biology or gene technology.
  • the specimen to be investigated is mounted for this purpose on a slide and brought into contact with a fluorescing label.
  • the substances present in the specimen and having an affinity with the label bind the label and can therefore be excited to emit fluorescent light.
  • Conclusions on the composition of the specimen can therefore be drawn by detecting the fluorescent light emitted by a specimen.
  • European Patent Application EP 0 947 823 A2 describes a device for detecting a fluorescent dye in specimens, the individual specimen points being arranged on a slide.
  • This slide is designed in the form of an optical conductor. Light is launched into the slide in order to excite the fluorescent dye at the individual specimen points. The fluorescent dye is then excited by the evanescent field formed at the interface between the slide and the specimen points.
  • the known device can be used to detect simultaneously the fluorescent light from a series of specimen points, for which purpose, for example, a linear photodiode array, or else a CCD row is used as detection device.
  • the device described in EP 0 947 823 A2 also permits simultaneous individual investigations of a multiplicity of specimen points in the case of which the fluorescent light from each individual specimen point is detected with the capability of assignment.
  • the present invention now proposes a very simple but extremely effective measure to prevent superimposition of the fluorescent light emitted by a plurality of specimen points at the location of a detection element, that is to say at the location of the measured value detection.
  • the spacing between the specimen points and the respectively assigned detection elements it is proposed for this purpose to select the spacing between the specimen points and the respectively assigned detection elements to be as small as possible so that the fluorescent light from the individual specimen points is detected in the manner of the production of a photographic contact print.
  • the invention it has been recognized that it is possible in this way to achieve very good results very easily.
  • the detection means could comprise a dedicated detection element for each specimen point.
  • each specimen point could be respectively assigned a plurality of detection elements.
  • the respective application is the determining factor in this case.
  • the detection elements In order to avoid superimposition of the fluorescent light from a plurality of specimen points at the location of a detection element, or of a plurality of respectively assigned detection elements, it is also possible to provide additional means within the scope of the present invention.
  • arranged for this purpose between the specimen points and the detection elements is at least one element which is transparent to fluorescent light emitted by a specimen point essentially in the direction of the assigned detection element, and totally reflects the fluorescent light emitted in the direction of adjacent specimen points, such that this fluorescent light is not detected.
  • Such an element can be implemented, for example, in the form of a glass plate which is arranged between the specimen points and the detection elements, provided that the slide is likewise of plate-shaped design and the detection elements are arranged in a plane parallel to the slide and opposite the surface of the slide which is provided with the specimen points.
  • the fluorescent light emitted at a steep angle by the specimen points in the direction of the detection elements can penetrate such a glass plate without hindrance, whereas the fluorescent light emitted in the direction of adjacent specimen points is totally reflected at the glass/air interface and deflected once more in the direction of the slide.
  • a glass plate has the advantage that it need not be specially adjusted. Moreover, it is also less expensive to produce and maintain.
  • the detection means of the arrangement according to the invention comprise a dedicated detection element for each specimen point.
  • the use of photodiode arrays or CCD chips has proved itself in practice.
  • the illumination of the specimen points with excitation light can also be implemented in various ways within the scope of the arrangement according to the invention for detecting fluorescent light from a plurality of specimen points.
  • An advantageous variant is so-called transillumination.
  • the slide is transparent at least to the excitation light.
  • the light source is then arranged on the side of the slide averted from the specimen points such that the specimen points are illuminated through the slide.
  • Another advantageous variant is total internal illumination by the slide.
  • the slide is designed in this case in the form of an optical conductor.
  • means are provided for launching the light of the light source into the optical conductor such that the specimen points are illuminated by the evanescent field formed at the interface between slide and specimen points.
  • a filter arrangement which is essentially transparent only to the fluorescent light to be detected is also additionally arranged between the specimen points and the detection elements.
  • a filter arrangement could comprise only a blocking filter for the excitation light emitted by the light source, in order to suppress interference caused thereby when detecting the fluorescent light emitted by the specimen points.
  • FIG. 1 shows the top view of a slide with specimen points
  • FIG. 2 shows the arrangement of a detection means over the slide illustrated in FIG. 1,
  • FIG. 3 shows the sectional illustration of an arrangement for detecting fluorescent light from a plurality of specimen points, in order to explain the beam path of the fluorescent light
  • FIG. 4 shows the sectional illustration of an arrangement according to the invention with transillumination for the specimen points
  • FIG. 5 shows the sectional illustration of an arrangement according to the invention with total internal illumination of the specimen points
  • FIG. 6 shows the arrangement, illustrated in FIG. 3, for the purpose of explaining the beam path of the fluorescent light
  • FIG. 7 shows a further arrangement according to the invention with mechanical barriers for the fluorescent light.
  • Specimen points 2 are arranged in an array on the slide 1 illustrated in FIG. 1 .
  • the specimen points 2 are microspots whose diameters are approximately 10 to 100 ⁇ m.
  • the individual specimen points are brought into contact with a fluorescing label such that they emit fluorescent light in the case of illumination with corresponding excitation light.
  • a plurality of specimen points here, all the specimen points 2 arranged on the slide 1 —are illuminated simultaneously with excitation light with the aid of the arrangement according to the invention for detecting fluorescent light.
  • the detection of the fluorescent light from the individual specimen points 2 is also performed simultaneously with the aid of the arrangement according to the invention, the detection means used therefor comprising a dedicated detection element for each specimen point.
  • the simultaneous detection of the fluorescent light from the individual specimen points 2 is performed with the aid of a photodiode array which is arranged over the arrangement of the specimen points 2 on the slide 1 such that each specimen point 2 is covered by exactly one photodiode 3 of the photodiode array.
  • FIG. 3 illustrates the beam path of the fluorescent light emitted by a specimen point 2 , and shows that the fraction of the fluorescent light emitted by a specimen point which is not detected by the detection element assigned to this specimen point becomes larger the larger the spacing d becomes between the specimen point and assigned detection element.
  • the specimen points 2 are covered in the arrangement illustrated in FIG. 3 by a cover glass 5 whose optical function is also explained in more detail in conjunction with FIG. 6 . Furthermore, there is arranged between the specimen points 2 and the detection elements 3 a filter 4 which is essentially transparent only to the fluorescent light emitted by the specimen points, and serves, in particular, as blocking filter for the excitation light.
  • the spacing between the slide 1 or the specimen points 2 and the respectively assigned detection elements 3 is selected to be as small as possible by arranging the detection elements 3 directly over the specimen points 2 , as it were. Only a filter 4 , which serves as blocking filter for the excitation light, is interposed here.
  • the specimen points 2 are illuminated from below through the slide 1 , which is transparent to the excitation light 6 , for which reason the arrangement illustrated in FIG. 4 is also denoted as a transillumination arrangement.
  • FIG. 5 shows a variant of the arrangement according to the invention with total internal illumination 7 of the specimen points 2 .
  • the slide 1 is designed here in the form of an optical conductor into which the light of a light source, for example a laser, is launched.
  • the illumination of the specimen points 2 is performed here via the evanescent field which forms at the interface between slide 1 and specimen points 2 .
  • the detection elements 3 are also arranged with the filter 4 directly over the specimen points 2 .
  • FIG. 6 illustrates the beam path of the fluorescent light, emitted uniformly in the entire solid angle, from a specimen point 2 , and the influence of the element 8 on this beam path.
  • the fluorescent light emitted in the direction of adjacent specimen points and the associated detection elements is totally reflected at the glass plate 8 , more precisely at the glass/air transition, such that this fraction of the fluorescent light is not detected by any detection element.
  • barriers 9 are also arranged between the individual specimen points 2 and the detection elements 3 . These barriers 9 delimit detection channels for the individual specimen points 2 and the assigned detection elements 3 , and in this way prevent superimposition of the fluorescent light from a plurality of specimen points 2 at the location of a detection element 3 .
  • the invention is based on the idea of illuminating a plurality of specimen points simultaneously and using an appropriate number of detection elements such as, for example, a photodiode array or a CCD chip to detect them simultaneously, in a fashion analogous to a contact print in photography.
  • This parallel read-out method is faster in principle than scanning methods.
  • crosstalk can be prevented when the light, which is emitted at a steep angle in the direction of an adjacent element, is totally reflected, for example at a glass/air interface.
  • mechanical barriers for avoiding crosstalk can be arranged between the individual detection elements.

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  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Optical Measuring Cells (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
US09/681,109 2000-01-28 2001-01-10 Arrangement for detecting fluorescent light from a plurality of sample points Expired - Lifetime US6603553B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10003754 2000-01-28
DE10003754 2000-01-28

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US6603553B1 true US6603553B1 (en) 2003-08-05

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US (1) US6603553B1 (de)
EP (1) EP1120643A3 (de)
JP (1) JP2001249077A (de)
DE (1) DE10024132B4 (de)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050043632A1 (en) * 2001-12-05 2005-02-24 Hans-Richard Kretschmer Assembly for the optical illumination of a plurality of samples
US20090069743A1 (en) * 2007-09-11 2009-03-12 Baxter International Inc. Infusion therapy sensor system
US20100051788A1 (en) * 2004-12-17 2010-03-04 Koninklijke Philips Electronics, N.V. Multi-spot investigation apparatus

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1347284B1 (de) * 2002-03-18 2008-01-09 ibidi GmbH Probenträger mit integrierter Optik
DE10225841A1 (de) * 2002-06-03 2003-12-11 Proteosys Ag Differentielle Anzeige von markierten Molekülen
JP6074624B2 (ja) * 2014-04-16 2017-02-08 学校法人北里研究所 生体試料中の個別多種及び／又は混合多種の疾患関連自己抗体の検出方法、キット及び検出装置

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5674698A (en) * 1992-09-14 1997-10-07 Sri International Up-converting reporters for biological and other assays using laser excitation techniques
EP0947823A2 (de) 1998-04-03 1999-10-06 Bodenseewerk Perkin-Elmer Gmbh Vorrichtung zum Nachweis eines Fluoreszenzfarbstoffs

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE59405534D1 (de) * 1993-07-02 1998-04-30 Inst Molekulare Biolog E V Probenträger und seine verwendung
US5759494A (en) * 1995-10-05 1998-06-02 Corning Incorporated Microplates which prevent optical cross-talk between wells
US5854684A (en) * 1996-09-26 1998-12-29 Sarnoff Corporation Massively parallel detection
DE19651935A1 (de) * 1996-12-14 1998-06-18 Ruckstuhl Thomas Detektionssystem für den optischen Nachweis von Molekülen
US5812272A (en) * 1997-01-30 1998-09-22 Hewlett-Packard Company Apparatus and method with tiled light source array for integrated assay sensing
DE19725050C2 (de) * 1997-06-13 1999-06-24 Fraunhofer Ges Forschung Anordnung zur Detektion biochemischer oder chemischer Substanzen mittels Fluoreszenzlichtanregung und Verfahren zu deren Herstellung
DE19808936A1 (de) * 1998-03-03 1999-09-16 Aventis Res & Tech Gmbh & Co Photodetektor und seine Verwendung
DE19810615A1 (de) * 1998-03-12 1999-09-16 Thomas Ruckstuhl Optische Anordnung zum Erfassen von Licht
DE19940751A1 (de) * 1998-08-28 2000-03-02 Febit Ferrarius Biotech Gmbh Lichtemissions-Detektionseinrichtung

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5674698A (en) * 1992-09-14 1997-10-07 Sri International Up-converting reporters for biological and other assays using laser excitation techniques
EP0947823A2 (de) 1998-04-03 1999-10-06 Bodenseewerk Perkin-Elmer Gmbh Vorrichtung zum Nachweis eines Fluoreszenzfarbstoffs

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050043632A1 (en) * 2001-12-05 2005-02-24 Hans-Richard Kretschmer Assembly for the optical illumination of a plurality of samples
US7227632B2 (en) 2001-12-05 2007-06-05 Siemens Aktiengesellschaft Assembly for the optical illumination of a plurality of samples
US20100051788A1 (en) * 2004-12-17 2010-03-04 Koninklijke Philips Electronics, N.V. Multi-spot investigation apparatus
US20090069743A1 (en) * 2007-09-11 2009-03-12 Baxter International Inc. Infusion therapy sensor system

Also Published As

Publication number Publication date
DE10024132A1 (de) 2001-08-09
JP2001249077A (ja) 2001-09-14
DE10024132B4 (de) 2007-03-22
EP1120643A2 (de) 2001-08-01
EP1120643A3 (de) 2002-03-13

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